1. Field of the invention
This invention relates to peptides useful in metalloproteinase detection and inhibition. Specifically, the invention relates to peptides derived from the sequence of type IV collagenase which correspond to domains of the enzyme which are involved in activation of the enzyme and interaction of the enzyme with its substrate. Antibodies recognizing the peptides are useful in enzyme detection. Specific peptides, identified by means of functional studies, constitute new classes of metalloproteinase inhibitors.
2. Background
The degradation of interstitial and basement membrane collagens is initiated by a specific class of metalloproteinase, the matrix metalloproteinases, also known as the collagenases (EC 3.4.24.7), which are secreted into the extracellular matrix in zymogen form. Members of this collagenase gene family include: the interstitial collagenases, which degrade collagen types I, II and III and have been characterized with respect to substrate specificity and requirements for activation (Stricklin, G. P., Jeffrey, J. J., Rosewit, W. T., and Eisen, A. Z., 1983, Biochemistry 22, 61-68; Goldberg, G. I., Wilhlem, S., Kronberger, A., Bauer, E. A., Grant, G. A., and Eisen, A. Z., 1986, J. Biol. Chem. 261, 6600`6605; Hasty, K. A., Jeffrey, J. J., Hibbs, M. S., and Welgus, H. G., 1987, J. Biol. Che, 262, 10048-1052; Fields, G. B., Van Wart, H. E., and Birkedal-Hansen, H., 1987, J. Biol. Chem. 262, 6221-6226; Grant, G. A., Eisen, A. Z., Marmer, B. L. Rosweit, W. T., and Goldberg, G. I., 1987, J. Biol. Chem. 262, 5886-5889); stromelysin, which degrades proteoglycans, glycoproteins, and the non-helical portions of collagenous molecules (Wilhelm, S. M., Collierm, I. E., Kronberger, A., Eisen, A. Z., Marmer, B. L., Grant, G. A., Bauer, E., and Goldberg, G. I., 1987, Proc. Natl. Acad. Sci. U.S.A. 84, 6725-6729; Whitman, S. E., Murphy, G., Angel, P., Rahmsforf, H.-J., SMith, B. J., Lyons, A., Harris, T. J. T., Reynolds. J. J., Herrlich, P. and Docherty, A. J. P., 1986, Biochem. J. 240, 913-916); and type IV collagenase, which degrades pepsin-resistant triple-helical type IV collagen and interstitial collagens (gelatin). Type IV collagenase has been identified in human tumor cells (Liotta, L. A., Kleinerman, J., Catanzaro, P., and Rynbrandt, D., 1977, J. Natl. Cancer Inst. 58, 1427-1439; Turpeenniemi-Hujanen, T., and Tryggvason, K., 1982, Int. J. Cancer 30p, 669-673; Liotta, L. A., Abe, S., Gehron-Robey, P., and Martin, G. R., 1979, Proc. Natl. Acad. Sci. U.S.A. 76 2268-2272; Liotta, L. A., Tryggvasson, K., Garbisa, S., Hart, I., Foltz, C. M., and Shafie, S., 1980, Nature, Lond., 284, 67-68; Collier, I. E., Wilhelm, S. M., Eisen, A. Z., Marmer, B. L., Grant, G. A., Seltzer, J. L., Kronberger, A , He., C., Bauer, E. A., and Goldberg, G. I., 1988, J. Biol. Chem. 263, 6579-6587), endothelial cells (Kalebic, T., Barbisa, S., Glaser, B., and Liotta, L. A., 1983, Science 221, 281-283), bone (Murphy, G., McAlpine, C. G., Poll, C. T., and Reynolds, J. J., 1985, Biochem. Biophys. Acta 831, 49-58), fibroblasts (Collier, I. E., Wilhelm, S. M., Eisen, A. Z., Marmer, B. L., Grant, G. A., Seltzer, J. L., Kronberger, A., He., C., Bauer, E. A., and Goldberg, G. I., 1988, J. Biol. Chem 263, 6579-6587), polymorphonuclear leukocytes (Uitto, V. J., Schwartx, D., and Veis, A., 1980, Eur. J. Biochem. 105, 409-417) and macrophages (Garbidsa, S., Ballin, M., Daga-Giordini, D., Fastelli, G., Naturale, M., Negro, A., Semenzato, G., and Liotta, L. A., 1986, J. Biol. Chem. 261, 2369-2375). This enzyme is a neutral metalloproteinase of 68 to 72 kilodaltons which is secreted in zymogen form (Liotta, L. A., Abe, S., Gehron-Robey, P., and Martin, G. R., 1979, Proc. Natl. Acad. Sci. U.S.A. 76, 2268-2272; Liotta, L. A., Tryggvassin, K., Garbisa, S., Gehron-Robey, P., and Abe, S., 1981, Biochemistry 20, 100-104; Salo, T., Liotta, L. A., and Tryggvsasson, K., 1983, J. Biol. Chem. 258, 3058-3063). In addition, several other members of this collagenase gene family have been described recently, including a second type of stromelysin (stromelysin-2), a 92 kilodalton form of type IV collagenase, and Putative Uterine Metalloproteinase (PUMP)-1, a low molecular weight uterine collagenase (Wilhelm, S. M., Collier, I. E., Marmer, B. L., Eisen, A. Z., Grant, G. A., and Goldberg, G. I., 1989, J. Biol. Chem. 264, 17213- 17221; Woessner, J. F. and Talpin, C. J., 1988, J. Biol. Chem. 263, 16918-16925).
The 70 kilodalton type IV collagenase has been closely linked to the metastatic potential of tumors in murine tumor models (Liotta, L. A., Tryggvasson, K., Garbisa, S., Hart, I., Foltz, C. M., and Shafie, S., 1980, Nature, Lond, 284, 67-68) and is augmented following H-ras oncogene induced genetic induction of the metastatic phenotype (Muschel, R., Williams, J. E., Lowy, D. R., and Liotta, L. A. 1985, Amer. J. Pathol 121, 1-8; Garbisa, S., Pozzatti, R., Muschel, R. J., Saffiotti, U., Ballin, M., Goldfarb, R. H., Khoury, G., and Liotta, L. A. 1987, Cancer Res. 47, 1523-1528). Trypsin treatment results in activation of the latent enzyme and a concomitant reduction in the molecular mass (Liotta, L. A., Tryggvasson, K., Garbisa, S., Gehron-Robery, P., and Abe, S., 1981, Biochemistry 20, 100-104). Organomercurial compounds have also been shown to activate this enzyme, and these are also associated with a reduction in the molecular mass (Collier, I. E., Wilhelm, S. M., Eisen, A. Z., Marmer, B. L., Grant, G. A., Seltzzer, J. L., Kronberger, A., He., C., Bauer, E. A., and Goldberg, G. I., 1988, J. Biol. Chem. 263, 6579-6587; Murphy, G., McAlpine, C. G., Poll, C. T., and Reynolds, J. J., 1985, Biochem. Biophys. Acta 831, 49-58). The activated enzyme cleaves type IV collagen to generate characteristic 1/4 amino-terminal and 3/4 carboxy-terminal fragments (Liotta, L. A., Tryggvasson, K., Garbis, S., Gehron-Robey, P. and Abe, S., 1981, Biochemistry 20, 100-104; Collier, I. E., Wilhelm, S. M., Eisen, A. Z., Marmer, B. L., Grant, G. A., Seltzer, J. L., Kronberger, A., He., C., Bauer, E. A., and Goldberg, G. I., 1988, J. Biol. Chem. 263, 6579-6587; Fessler, L. I., Duncan, K. G., Fessler, J. H., Salo, T., and Tryggvason, K., 1984, J. Biol. Chem. 259, 9783-9789). It has also been demonstrated that gelatinolytic activity is associated with this enzyme (Collier, I. E., Wilhelm, S. M., Eisen, A. Z., Marmer, B. L., Grant, G. A., Seltzer, J. L., Kronberger, A., He., C., Bauer, E. A., and Goldberg, G. I., 1988, J. Biol. Chem. 263, 6579-6587; Hoyhtya, M., Turpeenniemi-Hujanen, T., Stetler-Stevenson, W., Krutzsch, H., Tryggvason, K., and Liotta, L.A., 1988, FEBS Letters 233, 109-113; Murphy, G., McAlpine, C. G., Poll, C. T., and Reynolds, J. J., 1985, Biochem. Biophys. Acta 831, 49-58) as well as a type V collagenolytic activity (Collier, I. E., Wilhelm, S. M., Eisen, A. Z., Marmer, B. L., Grant, G. A., Seltzer, J. L., Kronberger, A., He., C., Bauer, E. A., and Goldberg, G. I., 1988, J. Biol. Chem. 263, 6579-6587; Murphy, G., McAlpine, C. G., Poll, C. T., and Reynolds, J. J., 1985, Biochem. Biophys. Acta 831, 49-58).
Type IV collagenase has been purified from human melanoma cells and sequence information on the intact protein amino terminus has been obtained as well as on tryptic and cyanogen bromide peptide fragments (Hoyhtya, M., Turpeenniemi-Hujanen, T., Stetler-Stevenson, W., Krutzsch, H., Tryggvason, K., and Liotta, L. A., 1988, FEBS Letters 233, 109-113). The sequence information demonstrates that type IV collagenase shows limited sequence homology to interstitial collagenase and stromelysin. A recent report has characterized a partial cDNA clone for a metalloproteinase secreted by H-ras-transformed human bronchial epithelial cells (Collier, I. E., Wilhelm, S. M., Eisen, A. Z., Marmer, B. L., Grant, G. A., Seltzer, J. L., Kronberger, A., He., C., Bauer, E. A., and Goldberg, G. I., 1988, J. Biol. Chem. 263, 6579-6587). The transformed bronchial epithelial enzyme is capable of specifically degrading type IV collagen, and the deduced amino acid sequence shows identity with that reported for tryptic and cyanogen bromide fragments of human tumor IV collagenase (Hoyhtya, M., Turpeenniemi-Hujanen, T., Stetler-Stevenson, W., Krutzsch, H., Tryggvason, K., and Liotta, L. A., 1988, FEBS Letters 233, 109-113). Thus, human melanoma cell type IV collagenase appears identical with the enzyme from H-ras-transformed bronchial epithelial cells, which is also found in fibroblasts (Collier, I. E., Wilhelm, S. M., Eisen, A. Z., Marmer, B. L., Grant, G. A., Seltzer, J. L., Kronberger, A., He., C., Bauer, E. A., and Goldberg, G. I., 1988, J. Biol. Chem. 263, 6579-6587) and bone cell explants (Murphy, G., McAlpine, C. G., Poll, C. T., and Reynolds, J. J., 1985, Biochem. Biophys. Acta 831, 49-58).